The present invention concerns a method of using a poly-GT element in the presence of an immediate-early gene product of.a large DNA virus to increase transcription of a recombinant gene in eukaryotic host cells. The poly-GT element exemplified herein is chemically synthesized (the prototype poly-GT element is depicted in Example 1 below). When inserted into an expression vector, the prototype poly-GT element comprises 21 repeating GT units. However, a wide variety of poly-GT elements, comprising repeating units of varying lengths will be recognized by those skilled in the art as suitable for use in the present invention, including chemically synthesized sequences or human genomic sequences. An enhancer element may be used along with the poly-GT element in the present invention to further increase such transcription. In some constructs, the poly-GT sequence is associated with the BK enhancer. The BK enhancer is a defined segment of DNA that consists of repeated sequences (the prototype BK enhancer and a variant BK enhancer are depicted in Example 18, below). However, a wide variety of BK enhancer variants, not all consisting of varying repeated sequences, are known in the art and suitable for use in the invention along with the poly-GT element.
The alternating sequence, poly (dT-dG).poly (dC-dA), is a highly repeated sequence in the eukaryotic genome and is capable of forming left-handed or Z-DNA. Hamada and Kakunaga. 1982, Nature 298:396-398; Hamada et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:6465-6469. For example, the human genome contains approximately 100,000 copies of 20 to 60 base pair (bp) tracts of poly (dT-dG) poly (dC-dA) randomly distributed. Hamada et al., 1984, Mol. Cell. Biol. 4:2610-2621. Hamada et al., 1984, Mol. Cell. Biol. 4:2622-2630, disclose that poly (dT-dG) poly (dC-dA) itself can act as an enhancer of the expression of chloramphenicol acetyltransferase (CAT) when the poly (dT-dG) poly (dC-dA) is linked to the SV40 early promoter and to the coding sequence for CAT. According to Hamada et al., supra, the enhancer-like activity of poly (dT-dG) poly (dC-dA) with the SV40 early promoter was much weaker than that of the SV40 enhancer with the SV40 early promoter, and, unlike many viral enhancers, the poly(dT-dG) poly (dC-dA) was equally active in monkey (CV-1) or human (HeLa) cells.
The BK enhancer sequences exemplified herein are obtained from BK virus, a human papovavirus that was first isolated from the urine of an immunosuppressed patient. BK virus is suspected of causing an unapparent childhood infection and is ubiquitous in the human population. Although BK virus grows optimally in human cells, the virus undergoes an abortive cycle in many non-primate cells, transforms rodent cells in vitro, and induces tumors in hamsters. BK virus is very similar to SV40 virus, but the enhancer sequences of these two papovaviruses, SV40 and BK, differ substantially in nucleotide sequence. The complete nucleotide sequence of BK virus (-5.2 kb) has been disclosed by Seif et al., 1979, Cell 18:963, and Yang and Wu, 1979, Science 206:456. Prototype BK virus is available from the American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md. 20852-1776, under the accession number ATCC VR-837. A restriction site and function map of prototype BK virus is presented in FIG. 1 of the accompanying drawings.
Enhancer elements are cis-acting elements that increase the level of transcription of an adjacent gene from its promoter in a fashion that is relatively independent of the position and orientation of the enhancer element. In fact, Khoury and Gruss, 1983, Cell 33:313, state that "the remarkable ability of enhancer sequences to function upstream from, within, or downstream from eukaryotic genes distinguishes them from classical promoter elements . . . " and suggest that certain experimental results indicate that "enhancers can act over considerable distances (perhaps &gt;10 kb)."
The present invention teaches a novel enhancer system, because the poly-GT element, when placed in combination with certain promoters, such as the adenovirus late promoter, functions as an enhancer only in the presence of a trans-acting early viral gene product, such as the E1A gene product. Other enhancer elements, such as the BK enhancer, may be used along with the poly-GT element in the present invention. The present invention specifically teaches an enhancer system for the adenovirus late promoter that results in unexpected increases in transcription only where there is a combination of a cis-acting poly-GT element with a trans-acting immediate-early gene product of a large DNA virus. Thus, a viral transacting protein is required to activate enhancer activity from the poly-GT element. This is in striking contrast to the enhancer system of Hamada et al, supra, where a poly-GT sequence alone was capable of enhancing expression of the CAT gene product using the SV40 early promoter. In the present invention, another enhancer element may be used along with the poly-GT element and the trans-acting viral gene product to further increase transcription and thus expression of a useful gene product.
When the BK enhancer is used along with the poly-GT element, still further increases in transcription result upon positioning the BK enhancer immediately upstream of (on the 5' side of) the "CCAAT" region of a eukaryotic promoter that is used in tandem with the BK enhancer to transcribe a DNA sequence encoding a useful substance. The CCAAT region or "immediate upstream region" or "-80 homology sequence" is a cis-acting upstream element that is a conserved region of nucleotides observed in promoters whose sequences for transcriptional activity have been dissected. The CCAAT region is found in many, but not all, promoters. In other promoters, equivalent cis-acting upstream sequences are found, including SP1 binding sites, ATF binding sites, the octa sequence, nuclear factor 1 binding sites (which may be the same binding site as the CCAAT transcription factor), the AP1 and AP2 homologies, glucocorticoid response elements, and heat shock response elements. Recently, a comprehensive compilation of currently known upstream sequences, including those just listed, has been published. Wingender, 1988, Nucl. Acids Res. 16:1879-1912. The CCAAT region equivalent in the adenovirus major la.te promoter is the upstream transcription factor (UTF) or major late transcription factor (MLTF) binding site (approximate nucleotides -50 to -65 upstream of the CAP site). The CCAAT sequence mediates the efficiency of transcription and, with few exceptions, cannot be deleted without decreasing promoter strength.
Enhancer elements have been identified in the DNA of a number of viruses, including murine polyomavirus, papilloma virus, adenovirus, retrovirus, hepatitis virus, cytomegalovirus, herpes virus, papovaviruses, such as SV40 and BK, and in many non-viral genes, such as within immunoglobulin gene introns. Enhancer elements may also be present in the DNA of a wide variety of other organisms. Enhancer elements often function differently in different host cells, and this cellular specificity can be due to differences in host gene products that interact with the enhancer element during gene expression.
The activity of enhancer elements can be affected by viral gene products present in the host cell. Velcich and Ziff, 1983, Cell 40:705; Borrelli et al., 1984, Nature 312:608; and Hen et al., 1985, Science 230:1391, disclose that the adenovirus-2 early region 1A (E1A) gene products repress activation of transcription induced by the SV40, polyoma virus, mouse immunoglobulin gene and adenovirus-2 E1A enhancers. This affect by E1A can be host cell dependent. Eukaryotic expression vectors that utilized enhancers to increase transcription of recombinant genes consequently were not expected to work better than vectors without enhancers in E1A-containing host cells. In striking contrast to the above-referenced prior art methods of using enhancers, co-pending application Ser. No. 07/129,028, filed on Dec. 4, 1987, attorney docket number X-6606A, teaches a method for using the BK virus enhancer element that involves using the E1A gene product or a similar immediate-early gene product of a large DNA virus to maximize gene expression. Thus, co-pending application Ser. No. 07/129,028 demonstrates. for the first time that the ability of the BK enhancer to promote transcription of DNA is increased in the presence of the E1A gene product of any adenovirus. It has now been unexpectedly found that a poly-GT element may be used along with the BK enhancer to further increase transcription of DNA in the presence of the E1A gene product of any adenovirus. When the adenovirus late promoter is used to drive transcription, the polyoGT element surprisingly does not itself enhance transcription, but is activated and does demonstrate enhancer activity only in the presence of the E1A gene product or a similar immediate-early gene product of a large DNA virus. Because the E1A gene product has been shown to repress activation of transcription by most enhancers tested, it was not expected that E1A would have the ability to activate an enhancer-like activity of a poly-GT element. The E1A gene product (actually, the E1A gene produces two products, which are collectively referred to herein as "the E1A gene product") is one such immediate-early gene product from adenovirus, a large DNA virus. The present invention encompasses the use of any immediate-early gene product of a large DNA virus or any retroviral protein product that functions similarly to the E1A gene product to increase the activity of a poly-GT element with or without another enhancer element, such as the BK enhancer. The herpes simplex virus ICP4 protein, described by DeLuca et al., 1985, Mol. Cell. Biol. 5:1997-2008, the pseudorabies virus IE protein, described by Feldman et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4952-4956, the cytomegalovirus IE protein described by Tevethia et al., 1987, Virology 161:276-285, and the E1B protein of adenovirus are all immediate-early gene products of large DNA viruses that have functions similar to the E1A protein. Therefore, the method of the present invention includes the use of the ICP4, IE, or E1B proteins, or any other similarly acting immediate-early gene product, either in the presence or absence of E1A protein, to produce or activate an enhancer-like activity from a poly-GT element. In addition, other trans-acting viral proteins, such as the TAT gene product of the HIV virus or other retroviral proteins, may be useful in the method to activate a poly-GT element.